Detection and quantification of specific nucleic acid sequences using PCR is useful in a large and diverse range of research and clinical applications. The first generation of PCR users performed end-point analysis by gel electrophoresis to obtain qualitative results. The advent of real-time PCR spawned a second generation that enabled quantification by monitoring the progression of amplification after each cycle using fluorescence probes. In real-time PCR, quantitative information can be obtained from the cycle threshold (CT), a point on the analogue fluorescence curve where the signal increases above background. External calibrators or normalization to endogenous controls can be required to estimate the concentration of an unknown. Imperfect amplification efficiencies affect CT values, which in-turn can limit the accuracy of this technique for absolute quantification.
Digital PCR combines limiting dilution, end-point PCR, and Poisson statistics to yield an absolute measure of nucleic acid concentration. In digital PCR, target DNA molecules are distributed across multiple replicate reactions at a level where some reactions have no template and others have one or more template copies present. After amplification to the terminal plateau phase of PCR, reactions containing one or more templates can yield positive end-points, whereas those without template can remain negative.
The TaqMan® assay can be used to detect the amplicons generated by PCR reactions. During TaqMan® amplification an internal probe can hybridize to an original template, or to an amplicon sequence generated using two primers used in a PCR. This internal probe can be labeled with two different dyes, e.g., a fluorophore and a quencher. When the two dyes are in close proximity, as is the case in an intact oligonucleotide probe, one of the dyes (e.g., TAMRA [N,N,N′,N′-tetramethyl-6-carboxyrhodamine]) can act as a quencher for a second fluorescent dye (e.g., FAM [5-carboxyfluorescein]) by absorbing at the FAM emission spectra. The 5′ exonuclease activity of Taq polymerase can degrade an internally hybridizing probe during the course of PCR. The degradation of the probe can lead to the separation of these two dyes in solution, with a subsequent increase in the level of fluorescence in the reaction mixture. The amount of fluorescence measured in a sample can be proportional to the amount of specific PCR product generated. Other assays that utilize a molecular beacon probe or a Scorpions™ Probe can be used to detect amplicons generated by PCR reactions.
PCR has been used to detect target polynucleotide sequences of interest in test samples. One example is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. SNPs are one of the most common types of genetic variation. A SNP can be a single base pair mutation at a specific locus, usually consisting of two alleles (where the rare allele frequency is >1%). SNPs can be involved in the etiology of many human diseases and are becoming of interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. SNPs can also provide a genetic fingerprint for use in identity testing.